Autoinducer-2 and bile salts induce c-di-GMP synthesis to repress the T3SS via a T3SS chaperone

Cyclic di-GMP (c-di-GMP) transduces extracellular stimuli into intracellular responses, coordinating a plethora of important biological processes. Low levels of c-di-GMP are often associated with highly virulent behavior that depends on the type III secretion system (T3SS) effectors encoded, whereas elevated levels of c-di-GMP lead to the repression of T3SSs. However, extracellular signals that modulate c-di-GMP metabolism to control T3SSs and c-di-GMP effectors that relay environmental stimuli to changes in T3SS activity remain largely obscure. Here, we show that the quorum sensing signal autoinducer-2 (AI-2) induces c-di-GMP synthesis via a GAPES1 domain-containing diguanylate cyclase (DGC) YeaJ to repress T3SS-1 gene expression in Salmonella enterica serovar Typhimurium. YeaJ homologs capable of sensing AI-2 are present in many other species belonging to Enterobacterales. We also reveal that taurocholate and taurodeoxycholate bind to the sensory domain of the DGC YedQ to induce intracellular accumulation of c-di-GMP, thus repressing the expression of T3SS-1 genes. Further, we find that c-di-GMP negatively controls the function of T3SSs through binding to the widely conserved CesD/SycD/LcrH family of T3SS chaperones. Our results support a model in which bacteria sense changes in population density and host-derived cues to regulate c-di-GMP synthesis, thereby modulating the activity of T3SSs via a c-di-GMP-responsive T3SS chaperone.

Growth curves and extracellular AI-2 activities of S. Typhimurium strains S. Typhimurium strains were grown at 37°C in LB medium with shaking (a) or in modified LB medium containing 0.3 M NaCl without agitation (b). At each time point during growth, cell density was recorded by measuring the optical density at 600 nm (solid line) and cell supernatants were collected, filtered, and measured for AI-2 activity by using the V. harveyi MM32 reporter assay (dashed line). Data are mean ± s.d. of three biological replicates. Source data are provided as a Source Data file.

Supplementary Fig. 4 Deletion of luxS did not alter intracellular levels of c-di-GMP in a ΔyeaJ
background LC-MS/MS measurements of cellular levels of c-di-GMP in S. Typhimurium strains. All strains were grown in LB broth at 37°C with shaking to an OD600 of 1.3 ( Supplementary Fig. 3a), and then 15 ml aliquots of the bacterial cultures were subjected to nucleotide extractions. Cellular levels of c-di-GMP were quantified by LC-MS/MS analysis. Data are mean ± s.d. of three biological replicates. Statistical significance was evaluated using the two-tailed unpaired Student's t-test. P values < 0.05 indicate significant differences. Source data are provided as a Source Data file.

Supplementary Fig. 5 AI-2-mediated regulation of biofilm formation and motility in S.
Typhimurium is dependent on the DGC YeaJ a Biofilm formation by S. Typhimurium strains was measured using a crystal violet staining method as described in Supplementary Fig. 1a. Data are mean ± s.e.m. of five independent experiments. b S.
Typhimurium strains were measured for swimming motility as described in Supplementary Fig. 1b. Data are mean ± s.e.m. of three independent experiments. a, b Statistical significance was determined by the two-tailed unpaired Student's t-test and p < 0.05 was considered to be statistically significant.
Source data are provided as a Source Data file.

high-confidence YeaJ orthologs from other genera
High-confidence YeaJ homologs were searched by BLASTP and further identified using InterProScan 5 against the Pfam 34.0 database (Supplementary Data 1). One representative sequence for each genus was randomly selected and multiple sequence alignments were performed with clustalX version 1.81.
The phylogenetic tree was reconstructed by using the maximum-likelihood method based on the Jones-Taylor-Thornton (JTT) model embedded in the MEGA7 software. The bacterial species to which YeaJ homologs belong are provided and the NCBI accession number for each YeaJ homolog is given in parentheses. The YeaJ homologs whose GAPES1 domains showed no detectable AI-2 binding activity are indicated in red. Phyletic distribution of the 19 GAPES1-containing proteins at the family level is Bacterial adherence (a) was measured by CFU counting and presented as the total number of cell-associated bacteria per well, and bacterial invasion (b) was expressed as a percentage of intracellular bacterial counts relative to the total adherent bacteria. Data are mean ± s.e.m. of three independent experiments. Statistical significance was determined by two-tailed Student's t-test. P < 0.05 indicates a statistically significant difference. Source data are provided as a Source Data file.

Supplementary Fig. 9 Competition assays in mice between S. Typhimurium strains
S. Typhimurium cultures grown in modified LB medium containing 0.3 M NaCl at 37°C without agitation to an OD600 of 1.1 were collected, washed and resuspended in PBS. BALB/c mice pre-treated with streptomycin were orally gavaged with a 1:1 mixture of two S. Typhimurium strains (5 × 10 8 CFU for each) carrying chloramphenicol-resistant (Cm r ) pBBR1MCS1 and kanamycin-resistant (Km r ) pKT100, respectively. Feces, cecum and small intestine tissues harvested at day 2 post infection were homogenized, serially diluted and plated on antibiotic selective LB agar plates for CFU enumeration. CI values were calculated as the ratios of the test strains carrying pBBR1MCS1 versus (vs) the control strains carrying pKT100 recovered from mice. Horizontal lines represent the geometric mean CI value for each group (n = 8 mice per group). Statistical significance was determined with a two-tailed Mann-Whitney U test. A p value less than 0.05 was considered to be statistically significant. Source data are provided as a Source Data file.